A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

The ydaO motif is an ATP-sensing riboswitch in Bacillus subtilis.

We report what is to our knowledge the first natural RNA that regulates gene expression in response to intracellular ATP. Using a biochemical screen, we found that several putative riboswitches bind ATP in vitro. The ydaO motif specifically bound ATP and regulated expression of endogenous and reporter genes in response to ATP concentrations in Bacillus subtilis. This discovery demonstrates a role for RNAs in regulating gene expression in response to energy balance in bacteria.

The glmS riboswitch integrates signals from activating and inhibitory metabolites in vivo.

The glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes. It is also a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS. The ligand concentration dependence of intracellular self-cleavage kinetics was measured for the first time in a yeast model system and unexpectedly revealed that this riboswitch is subject to inhibition as well as activation by hexose metabolites. Reporter gene experiments in Bacillus subtilis confirmed that this riboswitch integrates positive and negative chemical signals in its natural biological context. Contrary to the conventional view that a riboswitch responds to just a single cognate metabolite, our new model proposes that a single riboswitch integrates information from an array of chemical signals to modulate gene expression based on the overall metabolic state of the cell.

mRNA secondary structures fold sequentially but exchange rapidly in vivo.

RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts.

Determination of intracellular RNA folding rates using self-cleaving RNAs.

We have developed a system that relies on RNA self-cleavage to report quantitatively on assembly of RNA structures in vivo. Self-cleaving RNA sequences are inserted into mRNAs or snoRNAs and expressed in yeast under the control of a regulated promoter. Chimeric RNAs that contain self-cleaving ribozymes turn over faster than chimeric RNAs that contain a mutationally inactivated ribozyme by an amount that reflects the rate at which the ribozyme folds and self-cleaves. A key feature of this system is the choice of assay conditions that selectively monitor intracellular assembly and self-cleavage by suppressing further ribozyme activity during the analysis.

Resident and faculty perceptions of conflict of interest in medical education.

OBJECTIVE: To determine resident and faculty perceptions of the pharmaceutical industry's influence on medical education. DESIGN, SETTING, AND PARTICIPANTS: Anonymous survey of categorical residents and faculty in the department of medicine at a large, Midwestern, urban, independent academic medical center. MAIN RESULTS: Eighty-one residents (69.2%) and 196 faculty (75.7%) responded to the survey. Residents believed that a significantly higher percentage of primary care and subspecialist faculty receives industry income or gifts compared to faculty respondents. Many faculty, and to a significantly greater degree residents, indicated that income or gifts influence the teaching of both internal attending physicians and visiting faculty in a variety of educational settings. The majority of residents (61.7%) and faculty (62.2%) believed that annual income or gifts less than $10,000 could influence an attending physician's teaching. Most residents (65.4%) and faculty (74%) preferred that lecturers report all financial relationships with industry regardless of which relationships the lecturer believes are relevant. CONCLUSIONS: Most internal medicine residents and their faculty perceive that industry influences teaching in different educational settings, and want teachers to disclose all of their financial relationships with industry. This information may guide further development of policies and curricula addressing industry relationships within graduate medical education.